Proteins - Principle of food chemistry

INTRODUCTION
Proteins are polymers of some
21
different
amino
acids joined together by peptide
bonds. Because of the variety of side chains
that occur when these amino acids are linked
together, the different proteins may have dif-
ferent chemical properties and widely differ-
ent secondary and tertiary structures. The
various amino acids
joined
in a peptide chain
are shown in Figure
3-1.
The amino acids
are grouped on the basis of the chemical
nature of the side chains
(Krull
and Wall
1969).
The side chains may be polar or non-
polar. High levels of polar amino acid resi-
dues in a protein increase water solubility.
The most polar side chains are those of the
basic and acidic amino acids. These amino
acids are present at high levels in the soluble
albumins and globulins. In contrast, the wheat
proteins, gliadin and glutenin, have low levels
of polar side chains and are quite insoluble in

water. The acidic amino acids may also be
present in proteins in the form of their
amides, glutamine and asparagine. This
increases the nitrogen content of the protein.
Hydroxyl groups in the side chains may
become involved in ester linkages with phos-
phoric acid and phosphates. Sulfur amino
acids may form disulfide cross-links between
neighboring peptide chains or between dif-
ferent parts of the same chain. Proline and
hydroxyproline impose significant structural
limitations on the geometry of the peptide
chain.
Proteins occur in animal as well as vegeta-
ble products in important quantities. In the
developed countries, people obtain much of
their protein from animal products. In other
parts of the world, the major portion of
dietary protein is derived from plant prod-
ucts.
Many plant proteins are deficient in one
or more of the essential amino acids. The
protein content of some selected foods is
listed in Table
3-1.
AMINO ACID COMPOSITION
Amino acids joined together by peptide
bonds form the primary structure of
proteins.
The amino acid composition establishes the

nature of secondary and tertiary structures.
These, in turn, significantly influence the
functional properties of food proteins and
their behavior during processing. Of the 20
amino acids, only about half are essential for
human nutrition. The amounts of these essen-
tial amino acids present in a protein and their
availability determine the nutritional quality
of the protein. In general, animal proteins are
of higher quality than plant proteins. Plant
Proteins
CHAPTER
3
Figure 3-1 Component Amino Acids of Proteins
Joined by Peptide Bonds and Character of Side
Chains. Source: From Northern Regional Re-
search Laboratory, U.S. Department of Agricul-
ture.
proteins can be upgraded nutritionally by
judicious blending or by genetic modification
through plant breeding. The amino acid com-
position of some selected animal and vegeta-
ble proteins is given in Table
3—2.
Egg protein is one of the best quality pro-
teins and is considered to have a biological
value of 100. It is widely used as a standard,
and protein efficiency ratio (PER) values
sometimes use egg white as a standard.
Cereal proteins are generally deficient in

lysine and
threonine,
as indicated in Table
Table
3-1 Protein Content of Some Selected
Foods
Product Protein
(g/1
OO
g)
Meat:
beef 16.5
pork
10.2
Chicken
(light meat) 23.4
Fish:
haddock 18.3
cod
17.6
Milk
3.6
Egg
12.9
Wheat
13.3
Bread
8.7
Soybeans:
dry, raw 34.1

cooked
11.0
Peas
6.3
Beans:
dry, raw 22.3
cooked
7.8
Rice:
white,
raw 6.7
cooked
2.0
Cassava
1.6
Potato
2.0
Corn
10.0
3-3.
Soybean is a good source of
Iysine
but
is deficient in methionine. Cottonseed pro-
tein is deficient in lysine and peanut protein
in methionine and lysine. The protein of
potato although present in small quantity
(Table 3-1) is of excellent quality and is
equivalent to that of whole egg.
Table

3-3
Limiting Essential Amino Acids of
Some Grain Proteins
First Second
Limiting Limiting
Grain
Amino
Acid
Amino
Acid
Wheat Lysine Threonine
Corn Lysine Tryptophan
Rice Lysine Threonine
Sorghum Lysine Threonine
Millet Lysine Threonine
PROTEIN CLASSIFICATION
Proteins are complex molecules, and classi-
fication has been based mostly on solubility in
different solvents. Increasingly, however, as
more knowledge about molecular composi-
tion and structure is obtained, other criteria
are being used for classification. These
include behavior in the
ultracentrifuge
and
electrophoretic properties. Proteins are di-
vided into the following main groups: simple,
conjugated, and derived proteins.
Simple Proteins
Simple proteins yield only amino acids on

hydrolysis and include the following classes:
• Albumins. Soluble in neutral, salt-free
water. Usually these are proteins of rela-
tively low molecular weight. Examples
Table 3-2 Amino
AcJd
Content of Some Selected Foods (mg/g Total Nitrogen)
Amino
Acid
lsoleucine
Leucine
Lysine
Methlonine
Cystine
Phenylalanine
Tyroslne
Threonine
Valine
Arginine
Histidine
Alanine
Aspartic acid
Glutamic acid
Glycine
Proline
Serine
Meat
(Beef)
301
507

556
169
80
275
225
287
313
395
213
365
562
955
304
236
252
Milk
399
782
450
156
434
396
278
463
160
214
255
424
1151
144

514
342
Egg
393
551
436
210
152
358
260
320
428
381
152
370
601
796
207
260
478
Wheat
204
417
179
94
159
282
187
183
276

288
143
226
308
1866
245
621
281
Peas
267
425
470
57
70
287
171
254
294
595
143
255
685
1009
253
244
271
Com
230
783
167

120
97
305
239
225
303
262
170
471
392
1184
231
559
311
are egg albumin, lactalbumin, and serum
albumin in the whey proteins of milk,
leucosin of cereals, and legumelin in
legume seeds.
•
Globulins. Soluble in neutral salt solu-
tions and almost insoluble in water.
Examples are serum globulins and
(3-lac-
toglobulin in milk, myosin and actin in
meat, and glycinin in soybeans.
•
Glutelins. Soluble in very dilute acid or
base and insoluble in neutral solvents.
These proteins occur in cereals, such as
glutenin in wheat and oryzenin in rice.

•
Prolamins.
Soluble in 50 to 90 percent
ethanol and insoluble in water. These
proteins have large amounts of proline
and glutamic acid and occur in cereals.
Examples are zein in corn, gliadin in
wheat, and hordein in barley.
•
Scleroproteins. Insoluble in water and
neutral solvents and resistant to enzymic
hydrolysis. These are fibrous proteins
serving structural and binding purposes.
Collagen of muscle tissue is included in
this group, as is gelatin, which is derived
from it. Other examples include elastin,
a component of tendons, and keratin, a
component of hair and
hoofs.
•
Histories. Basic proteins, as defined by
their high content of lysine and arginine.
Soluble in water and precipitated by
ammonia.
•
Protamines. Strongly basic proteins of
low molecular weight (4,000 to 8,000).
They are rich in arginine. Examples are
clupein from herring and scombrin from
mackerel.

Conjugated
Proteins
Conjugated proteins contain an amino
acid part combined with a nonprotein mate-
rial such as a
lipid,
nucleic acid, or carbohy-
drate.
Some of the major conjugated
proteins are as follows:
•
Phosphoproteins. An important group
that includes many major food proteins.
Phosphate groups are linked to the
hydroxyl groups of serine and threonine.
This group includes casein of milk and
the phosphoproteins of egg yolk.
•
Lipoproteins.
These are combinations of
lipids with protein and have excellent
emulsifying capacity. Lipoproteins occur
in milk and egg yolk.
•
Nucleoproteins. These are combinations
of nucleic acids with protein. These
compounds are found in cell nuclei.
•
Glycoproteins.
These are combinations

of carbohydrates with protein. Usually
the amount of carbohydrate is small, but
some glycoproteins have carbohydrate
contents of 8 to 20 percent. An example
of such a mucoprotein is ovomucin of
egg white.
•
Chromopmteins.
These are proteins with
a colored prosthetic group. There are
many compounds of this type, including
hemoglobin and myoglobin, chlorophyll,
and flavoproteins.
Derived Proteins
These are compounds obtained by chemi-
cal or enzymatic methods and are divided
into primary and secondary derivatives, de-
pending on the extent of change that has
taken place. Primary derivatives are slightly
modified and are insoluble in water; rennet-
coagulated casein is an example of a primary
derivative. Secondary derivatives are more
extensively changed and include proteoses,
peptones, and peptides. The difference
between these breakdown products is in size
and solubility. All are soluble in water and
not coagulated by heat, but proteoses can be
precipitated with saturated ammonium
sul-
fate solution. Peptides contain two or more

amino acid residues. These breakdown prod-
ucts are formed during the processing of
many foods, for example, during ripening of
cheese.
PROTEIN STRUCTURE
Proteins are macromolecules with different
levels of structural organization. The primary
structure of proteins relates to the peptide
bonds between component amino acids and
also to the amino acid sequence in the mole-
cule.
Researchers have elucidated the amino
acid sequence in many proteins. For exam-
ple,
the amino acid composition and se-
quence for several milk proteins is now well
established (Swaisgood 1982).
Some
proteolytic
enzymes have quite spe-
cific actions; they attack only a limited num-
ber of bonds, involving only particular amino
acid residues in a particular sequence. This
may lead to the accumulation of well-defined
peptides during some enzymic proteolytic
reactions in foods.
The secondary structure of proteins in-
volves folding the primary structure. Hydro-
gen bonds between amide nitrogen and car-
bonyl

oxygen are the major stabilizing force.
These bonds may be formed between differ-
ent areas of the same polypeptide chain or
between adjacent chains. In aqueous media,
the hydrogen bonds may be less significant,
and van der
Waals
forces and hydrophobic
interaction between apolar side chains may
contribute to the stability of the secondary
structure. The secondary structure may be
either the
oc-helix
or the sheet structure, as
shown in Figure 3-2. The helical structures
are stabilized by intramolecular hydrogen
bonds, the sheet structures by
intermolecular
hydrogen bonds. The requirements for maxi-
mum stability of the helix structure were
established by Pauling et
al.
(1951). The
helix model involves a translation of 0.54 nm
per turn along the central axis. A complete
turn is made for every 3.6 amino acid resi-
dues.
Proteins do not necessarily have to
occur in a complete
a-helix

configuration;
rather, only parts of the peptide chains may
be helical, with other areas of the chain in a
more or less unordered configuration. Pro-
teins with a-helix structure may be either
globular or fibrous. In the parallel sheet
structure, the polypeptide chains are almost
fully extended and can form hydrogen bonds
between adjacent chains. Such structures are
generally insoluble in aqueous solvents and
are fibrous in nature.
The tertiary structure of proteins involves a
pattern of folding of the chains into a com-
pact unit that is stabilized by hydrogen bonds,
van der Waals forces,
disulfide
bridges, and
hydrophobic interactions. The tertiary struc-
ture results in the formation of a tightly
packed unit with most of the polar amino acid
residues located on the outside and hydrated.
This leaves the internal part with most of the
apolar side chains and virtually no hydration.
Certain amino acids, such as proline, disrupt
the a-helix, and this causes fold regions with
random structure (Kinsella 1982). The nature
of the tertiary structure varies among proteins
as does the ratio of a-helix and random coil.
Insulin is loosely folded, and its tertiary struc-
ture is stabilized by disulfide bridges.

Lyso-
zyme and glycinin have disulfide bridges but
are compactly folded.
Large molecules of molecular weights
above about 50,000 may form quaternary
structures by association of subunits. These
structures may be stabilized by hydrogen
bonds, disulfide bridges, and hydrophobic
interactions. The bond energies involved in
Figure 3-2 Secondary Structures of Proteins, (A) Alpha Helix, (B)
Antiparallel
Sheet
3rd
turn
2nd
turn
1st
turn
Rise per
residue
forming these structures are listed in Table
3-4.
The term subunit denotes a protein chain
possessing an internal covalent and noncova-
lent
structure that is capable of joining with
other similar subunits through noncovalent
forces or disulfide bonds to form an
oligo-
meric macromolecule (Stanley and Yada

1992).
Many food proteins are oligomeric
and consist of a number of subunits, usually
2 or 4, but occasionally as many as 24. A list-
ing of some oligomeric food proteins is
given in Table
3-5.
The subunits of proteins
are held together by various types of bonds:
electrostatic bonds involving carboxyl,
amino, imidazole, and
guanido
groups; hy-
drogen bonds involving hydroxyl, amide,
and phenol groups; hydrophobic bonds in-
volving long-chain aliphatic residues or aro-
matic groups; and covalent disulfide bonds
involving cystine residues. Hydrophobic
bonds are not true bonds but have been
described as interactions of nonpolar groups.
These nonpolar groups or areas have a ten-
dency to orient themselves to the interior of
the protein molecule. This tendency depends
on the relative number of nonpolar amino
Table
3-4
Bond Energies of the Bonds Involved
in Protein Structure
Bond
Energy*

Bond
(kcal/mole)
Covalent C-C 83
Covalent S-S 50
Hydrogen bond 3-7
Ionic electrostatic bond 3-7
Hydrophobic bond 3-5
Van der
Waals
bond
1
-2
These refer to free energy required to break the
bonds: in the case of a hydrophobic bond, the free
energy required to unfold a nonpolar side chain from
the interior of the molecule into the aqueous medium.
acid residues and their location in the peptide
chain. Many food proteins, especially plant
storage proteins, are highly
hydrophobic—so
much so that not all of the hydrophobic areas
can be oriented toward the inside and have to
be located on the surface. This is a possible
factor in subunits association and in some
cases may result in aggregation. The hydro-
phobicity values of some food proteins as
reported by Stanley and Yada
(1992)
are
listed in Table 3-6.

The well-defined secondary, tertiary, and
quaternary structures are thought to arise
directly from the primary structure. This
means that a given combination of amino
acids will automatically assume the type of
structure that is most stable and possible
given the considerations described by Paul-
ing
etal.
(1951).
Table 3-5 Oligomeric Food Proteins
Molecular
Protein
Weight
(d) Subunits
Lactoglobulin 35,000 2
Hemoglobin 64,500 4
Avidin 68,300 4
Lipoxygenase 108,000 2
Tyrosinase 128,000 4
Lactate 140,000 4
dehydrogenase
7S soy protein 200,000 9
Invertase 210,000 4
Catalase 232,000 4
Collagen 300,000 3
11S
soy protein 350,000
12
Legumin 360,000 6

Myosin 475,000 6
Source:
Reprinted with permission from
D.W.
Stanley
and R.Y. Yada, Thermal Reactions in Food Protein Sys-
tems,
Physical Chemistry
of
Foods,
H.G.
Schwartzberg
and R.H. Hartel,
eds.,
p. 676, 1992, by courtesy of Mar-
cel
Dekker,
Inc.
DENATURATION
Denaturation
is a process that changes the
molecular structure without breaking any of
the peptide bonds of a protein. The process is
peculiar to proteins and affects different pro-
teins to different degrees, depending on the
structure of a protein. Denaturation can be
brought about by a variety of agents, of
which the most important are heat, pH, salts,
and surface effects. Considering the com-
plexity of many food systems, it is not sur-

prising that denaturation is a complex pro-
cess that cannot easily be described in simple
terms.
Denaturation usually involves loss of
biological activity and significant changes in
some physical or functional properties such
as solubility. The destruction of enzyme
activity by heat is an important operation in
food processing. In most cases, denaturation
is nonreversible; however, there are some
Table
3-6
Hydrophobicity Values of Some Food
Proteins
Hydrophobicity
Protein cal/residue
Gliadin
1300
Bovine
serum albumin
1120-1000
oc-Lactalbumin
1050
(3-Lactoglobulin
1050
Actin
1000
Ovalbumin
980
Collagen

880
Myosin
880
Casein
725
Whey
protein 387
Gluten
349
Source:
Reprinted with permission from
D.W.
Stanley
and
R.Y.
Yada,
Thermal Reactions in Food Protein Sys-
tems,
Physical Chemistry
of
Foods,
H.G.
Schwartzberg
and
R.H.
Hartel,
eds., p. 677,
1992,
by courtesy of Mar-
cel

Dekker, Inc.
exceptions, such as the recovery of some
types of enzyme activity after heating. Heat
denaturation is sometimes
desirable—for
example, the denaturation of whey proteins
for the production of milk powder used in
baking. The relationship among temperature,
heating time, and the extent of whey protein
denaturation in skim milk is demonstrated in
Figure 3-3 (Harland et
al.
1952).
The proteins of egg white are readily
dena-
tured by heat and by surface forces when egg
white is whipped to a foam. Meat proteins
are denatured in the temperature range 57 to
75
0
C,
which has a profound effect on tex-
ture,
water holding capacity, and shrinkage.
Denaturation may sometimes result in the
flocculation of globular proteins but may
also lead to the formation of gels. Foods may
be denatured, and their proteins destabilized,
during freezing and frozen storage. Fish pro-
teins are particularly susceptible to destabili-

zation. After freezing, fish may become
tough and rubbery and lose moisture. The
caseinate micelles of milk, which are quite
stable to heat, may be destabilized by freez-
ing.
On frozen storage of milk, the stability
of the caseinate progressively decreases, and
this may lead to complete coagulation.
Protein denaturation and coagulation are
aspects of heat stability that can be related to
the amino acid composition and sequence of
the protein. Denaturation can be defined as a
major change in the native structure that
does not involve alteration of the amino acid
sequence. The effect of heat usually involves
a change in the tertiary structure, leading to a
less ordered arrangement of the polypeptide
chains.
The temperature range in which
denaturation and coagulation of most pro-
teins take place is about 55 to
75
0
C,
as indi-
cated in Table 3-7. There are some notable
exceptions to this general pattern. Casein and
gelatin are examples of proteins that can be
boiled without apparent change in stability.
The exceptional stability of casein makes it

possible to boil, sterilize, and concentrate
milk, without coagulation. The reasons for
this exceptional stability have been discussed
by Kirchmeier (1962). In the first place,
restricted formation of disulfide bonds due to
low content of cystine and cysteine results in
increased stability. The relationship between
coagulation temperature as a measure of sta-
Figure 3-3 Time-Temperature Relationships for the Heat Denaturation of Whey Proteins in Skim
Milk. Source: From H.A. Harland, S.T. Coulter, and R. Jenness, The Effects of Various Steps in the
Manufacture on the Extent of Serum Protein Denaturation in Nonfat Dry Milk Solids. /. Dairy ScL
35:
363-368, 1952.
TCNPCKATURf
PER
CENT
DeNATUMTK)N
TIME
OF
HCATINO
IN
MINUTCS
bility and sulfur amino acid content is shown
in Tables 3-7 and 3-8. Peptides, which are
low in these particular amino acids, are less
likely to become involved in the type of
sulf-
hydryl
agglomeration shown in Figure 3-4.
Casein, with its extremely low content of

sulfur amino acids, exemplifies this behav-
ior. The heat stability of casein is also
explained by the restraints against forming a
folded tertiary structure. These restraints are
due to the relatively high content of proline
and hydroxyproline in the heat stable pro-
teins (Table 3-9). In a peptide chain free of
proline, the possibility of forming inter- and
intramolecular hydrogen bonds is better than
in a chain containing many proline residues
(Figure 3-5). These considerations show
how amino acid composition directly relates
to secondary and tertiary structure of pro-
teins;
these structures are, in turn, responsi-
ble for some of the physical properties of the
protein and the food of which it is a part.
NONENZYMIC BROWNING
The nonenzymic browning or Maillard
reaction is of great importance in food man-
ufacturing and its results can be either desir-
Table
3-7 Heat Coagulation Temperatures of
Some Albumins and Globulins and Casein
Coagulation
Protein
Temp.
(
0
C)

Egg albumin 56
Serum albumin (bovine) 67
Milk albumin (bovine) 72
Legumelin (pea) 60
Serum globulin (human) 75
p-Lactoglobulin
(bovine) 70-75
Fibrinogen (human) 56-64
Myosin (rabbit) 47-56
Casein (bovine) 160-200
able or undesirable. For example, the brown
crust formation on bread is desirable; the
brown discoloration of evaporated and steril-
ized milk is undesirable. For products in
which the browning reaction is favorable, the
resulting color and flavor characteristics are
generally experienced as pleasant. In other
products, color and flavor may become quite
unpleasant.
The browning reaction can be defined as the
sequence of events that begins with the reac-
tion of the amino group of amino acids, pep-
tides,
or proteins with a glycosidic hydroxyl
group of sugars; the sequence terminates with
the formation of brown nitrogenous polymers
or melanoidins (Ellis 1959).
The reaction velocity and pattern are influ-
enced by the nature of the reacting amino
acid or protein and the carbohydrate. This

means that each kind of food may show a
different browning pattern. Generally,
Iysine
is the most reactive amino acid because of
the free £-amino group. Since lysine is the
limiting essential amino acid in many food
proteins, its destruction can substantially
reduce the nutritional value of the protein.
Foods that are rich in reducing sugars are
very reactive, and this explains why lysine in
milk is destroyed more easily than in other
Table
3-8
Cysteine and Cystine Content of
Some Proteins (g Amino
Acid/100
g Protein)
Cysteine Cystine
Protein
(%) (%)
Egg albumin
1.4
0.5
Serum albumin 0.3 5.7
(bovine)
Milk albumin 6.4 —
p-Lactoglobulin 1.1 2.3
Fibrinogen 0.4 2.3
Casein — 0.3
Figure

3-4
Reactions Involved in
Sulfhydryl
Polymerization of Proteins.
Source:
From O.
Kirchmeier,
The
Physical-Chemical
Causes of
the Heat Stability of Milk Proteins,
Milchwis-
senschaft
(German), Vol. 17, pp.
408-412,
1962.
foods (Figure 3-6). Other factors that influ-
ence the browning reaction are temperature,
pH,
moisture level, oxygen, metals, phos-
phates, sulfur dioxide, and other inhibitors.
The browning reaction involves a number
of steps. An outline of the total pathway of
melanoidin
formation has been given by
Hodge (1953) and is shown in Figure 3-7.
According to Hurst
(1972),
the following
five steps are involved in the process:

1.
The production of an
Af-substituted
glycosylamine from an aldose or
ketose reacting with a primary amino
group of an amino acid, peptide, or
protein.
2.
Rearrangement of the glycosylamine
by an Amadori rearrangement type of
reaction to yield an aldoseamine or
ketoseamine.
3.
A second rearrangement of the ketose-
amine with a second mole of aldose to
form a diketoseamine, or the reaction
Table
3-9 Amino Acid Composition of Serum
Albumin,
Casein,
and Gelatin (g Amino
Acid/100
g Protein)
Amino
Acid
Glycine
Alanine
Valine
Leucine
isoleucine

Serine
Threonine
Cystine
1/2
Methionine
Phenylalanine
Tyrosine
Proline
Hydroxyproline
Aspartic
acid
Glutamic
acid
Lysine
Arginine
Histidine
Serum
Albumin
1.8
6.3
5.9
12.3
2.6
4.2
5.8
6.0
0.8
6.6
5.1
4.8

10.9
16.5
12.8
5.9
4.0
Casein
1.9
3.1
6.8
9.2
5.6
5.3
4.4
0.3
1.8
5.3
5.7
13.5
7.6
24.5
8.9
3.3
3.8
Gelatin
27.5
11.0
2.6
3.3
1.7
4.2

2.2
0.0
0.9
2.2
0.3
16.4
14.1
6.7
11.4
4.5
8.8
0.8
of an aldoseamine with a second mole
of amino acid to yield a diamino sugar.
4.
Degradation of the amino sugars with
loss of one or more molecules of water
to give amino or nonamino com-
pounds.
5.
Condensation of the compounds
formed in Step 4 with each other or
with amino compounds to form brown
pigments and
polymers.
The formation of glycosylamines from the
reaction of amino groups and sugars is
reversible (Figure 3-8) and the equilibrium
is highly dependent on the moisture level.
The mechanism as shown is thought to

involve addition of the amine to the carbonyl
group of the open-chain form of the sugar,
elimination of a molecule of water, and clo-
sure of the ring. The rate is high at low water
content; this explains the ease of browning in
dried and concentrated foods.
The
Amadori
rearrangement of the glyco-
sylamines involves the presence of an acid
catalyst and leads to the formation of ketose-
amine or
1-amino-1-deoxyketose
according
Figure 3-5 Effect of Proline Residues on Possible Hydrogen Bond Formation in Peptide Chains. (A)
Proline-free
chain; (B) proline-containing chain; (C) hydrogen bond formation in
proline-free
and pro-
line-containing chains.
Source:
From O. Kirchmeier, The Physical-Chemical Causes of the Heat Sta-
bility of Milk Proteins,
Milchwissenschaft
(German), Vol. 17, pp. 408-412, 1962.
A
B
C
to the scheme shown in Figure 3-9. In the
reaction of

D-glucose
with glycine, the amino
acid reacts as the catalyst and the compound
produced is
1-deoxy-l-glycino-p-D-fructose
(Figure 3-10). The
ketoseamines
are rela-
tively stable compounds, which are formed in
maximum yield in systems with 18 percent
water content. A second type of rearangement
reaction is the Heyns rearrangement, which is
an alternative to the Amadori rearrangement
and leads to the same type of transformation.
The mechanism of the Amadori rearrange-
ment (Figure 3-9) involves protonation of the
nitrogen atom at carbon
1.
The Heyns rear-
rangement (Figure
3-11)
involves protona-
tion of the oxygen at carbon
6.
Secondary reactions lead to the formation
of
diketoseamines
and diamino sugars. The
formation of these compounds involves com-
plex reactions and, in contrast to the forma-

tion of the primary products, does not occur
on a
mole-for-mole
basis.
In Step 4, the ketoseamines are decom-
posed by
1,2-enolization
or
2,3-enolization.
The former pathway appears to be the more
important one for the formation of brown
color, whereas the latter results in the forma-
tion of flavor products. According to Hurst
(1972),
the 1,2-enolization pathway appears
mainly to lead to browning but also contrib-
utes to formation of off-flavors through
hydroxymethylfurfural,
which may be a fac-
Figure
3-6
Loss of Lysine Occurring as a Result of Heating of Several Foods. Source: From J. Adrian,
The
Maillard
Reaction. IV. Study on the Behavior of Some Amino Acids During Roasting of Proteina-
ceous Foods, Ann.
Nutr.
Aliment.
(French), Vol. 21, pp. 129-147, 1967.
Heating

at 150° (minutes)
Cotton
Peanut
Wheat
Milk
Loss
of lysine
tor in causing off-flavors in stored, over-
heated, or dehydrated food products. The
mechanism of this reaction is shown in Fig-
ure 3-12 (Hurst 1972). The ketoseamine (1)
is protonated in acid medium to yield (2).
This is changed in a reversible reaction into
the
1,2-enolamine
(3) and this is assisted by
the N
substituent
on carbon
1.
The following
steps involve the
p-elimination
of the hy-
droxyl group on carbon 3. In (4) the
enola-
mine is in the free base form and converts to
the Schiff base (5). The Schiff base may
Figure 3-7 Reaction Pattern of the Formation of Melanoidins from Aldose Sugars and Amino Com-
pounds. Source: From

I.E.
Hodge, Chemistry of Browning Reactions in Model Systems, Agr. Food
Chem.,
Vol. 1, pp. 928-943, 1953.
Figure 3-8 Reversible Formation of Glycosylamines in the Browning Reaction. Source: From D.T.
Hurst, Recent Development in the Study
ofNonenzymic
Browning and Its Inhibition by Sulpher Diox-
ide,
BFMIRA Scientific and Technical Surveys, No. 75, Leatherhead, England, 1972.
Amadori Rearrangement
a-D-Glucopyrano-
sylamine
1-Amino-l-deoxy-a-
D-fructopyranose
Figure 3-9 Amadori Rearrangement. Source: From MJ.
Kort,
Reactions of Free Sugars with Aqueous
Ammonia, Adv. Carbohydrate Chem.
Biochem.,
Vol. 25, pp
311-349,
1970.
Figure 3-10 Structure of
1-Deoxy-l-Glycino-p-
D-Fructose
undergo hydrolysis and form the
enolform
(7)
of

3-deoxyosulose
(8). In another step the
Schiff
base (5) may lose a proton and the
hydroxyl from carbon 4 to yield a new Schiff
base (6). Both this compound and the 3-deox-
yosulose may be transformed into an unsatur-
ated osulose (9), and by elimination of a
proton and a hydroxyl group, hydroxymeth-
y!furfural
(10)
is formed.
Following the production of
1,2-enol
forms
of aldose and ketose amines, a series of deg-
radations and condensations results in the for-
mation of melanoidins. The
oc-p-dicarbonyl
compounds enter into
aldol
type condensa-
tions,
which lead to the formation of poly-
mers,
initially of small size, highly hydrated,
and in colloidal form. These initial products
of condensation are fluorescent, and continu-
ation of the reaction results in the formation
of the brown melanoidins. These polymers

are of nondistinct composition and contain
cr-D-Fructopyrano-
sylamine
2-Amino-2-deoxy-o?-
D-glucopyranose
Figure 3-11 Heyns Rearrangement. Source: From MJ. Kort, Reactions of Free Sugars with Aqueous
Ammonia, Adv. Carbohydrate Chem.
Biochem.,
Vol. 25, pp.
311-349,
1970.
varying levels of nitrogen. The composition
varies with the nature of the reaction partners,
pH,
temperature, and other conditions.
The flavors produced by the Maillard reac-
tion also vary widely. In some cases, the fla-
vor is reminiscent of caramelization. The
Strecker degradation of
a-amino
acids is a
reaction that also significantly contributes to
the formation of flavor compounds. The
dicarbonyl compounds formed in the previ-
ously described schemes react in the follow-
ing manner with a-amino
acids:
Figure 3-12
1,2-Enolization
Mechanism of the Browning Reaction. Source: From D.T. Hurst, Recent

Developments in the Study of Nonenzymic Browning and Its Inhibition by Sulphur Dioxide,
BFMIRA Scientific and Technical Surveys, No. 75, Leatherhead, England, 1972.
The amino acid is converted into an aldehyde
with one less carbon atom
(Schonberg
and
Moubacher 1952). Some of the compounds
of browning flavor have been described by
Hodge et
al.
(1972). Corny, nutty, bready,
and crackery aroma compounds consist of
planar unsaturated heterocyclic compounds
with one or two nitrogen atoms in the ring.
Other important members of this group are
partially saturated
Af-heterocyclics
with
alkyl
or acetyl group
substituents.
Compounds that
contribute to pungent, burnt aromas are
listed in Table 3-10. These are mostly vici-
nal
polycarbonyl
compounds and
oc,p-unsat-
urated aldehydes. They condense rapidly to
form melanoidins. The

Strecker
degradation
aldehydes contribute to the aroma of bread,
peanuts, cocoa, and other roasted foods. Al-
though acetic, phenylacetic, isobutyric, and
isovaleric
aldehydes are prominent in the
aromas of bread, malt, peanuts, and cocoa,
they are not really characteristic of these
foods (Hodge et al. 1972).
A somewhat different mechanism for the
browning reaction has been proposed by
Burton and
McWeeney
(1964) and is shown
in Figure
3-13.
After formation of the
aldo-
sylamine, dehydration reactions result in the
production of 4- to 6-membered ring com-
pounds. When the reaction proceeds under
conditions of moderate heating, fluorescent
nitrogenous compounds are formed. These
react rapidly with glycine to yield melanoi-
dins.
The influence of reaction components and
reaction conditions results in a wide variety
of reaction patterns. Many of these condi-
tions are interdependent. Increasing tempera-

ture results in a rapidly increasing rate of
browning; not only reaction rate, but also the
pattern of the reaction may change with tem-
perature. In model systems, the rate of
browning increases two to three times for
each 10° rise in temperature. In foods con-
taining fructose, the increase may be 5 to 10
times for each 10° rise. At high sugar con-
tents,
the rate may be even more rapid. Tem-
perature also affects the composition of the
pigment formed. At higher temperatures, the
carbon content of the pigment increases and
more pigment is formed per mole of carbon
dioxide released. Color intensity of the pig-
ment increases with increasing temperature.
The effect of temperature on the reaction rate
of
D-glucose
with
DL-leucine
is illustrated
in Figure
3-14.
In the Maillard reaction, the basic amino
group disappears; therefore, the initial pH or
the presence of a buffer has an important
effect on the reaction. The browning reaction
is slowed down by decreasing pH, and the
browning reaction can be said to be

self-
inhibitory since the pH decreases with the
loss of the basic amino group. The effect of
pH on the reaction rate of D-glucose with
DL-leucine is demonstrated in Figure
3-15.
The effect of pH on the browning reaction is
highly dependent on moisture content. When
a large amount of water is present, most of
the browning is caused by
caramelization,
but at low water levels and at pH greater than
6, the Maillard reaction is predominant.
The nature of the sugars in a
nonenzymic
browning reaction determines their reactiv-
ity. Reactivity is related to their conforma-
tional stability or to the amount of open-
chain structure present in solution. Pentoses
are more reactive than hexoses, and hexoses
more than reducing disaccharides. Nonre-
ducing disaccharides only react after hydrol-
ysis has taken place. The order of reactivity
of some of the aldohexoses is: mannose is
more reactive than galactose, which is more
reactive than glucose.
The effect of the type of amino acid can be
summarized as follows. In the
a-amino
acid

Figure
3-13
Proposed
Browning
Reaction Mechanism
According
to
Burton
and
McWeeney.
Source:
From
H.S.
Burton
and DJ.
McWeeney,
Non-Enzymatic
Browning:
Routes
to the
Production
of
MeI-
anoidins
from
Aldoses
and
Amino
Compounds,
Chem.

Ind.,
Vol.
11,
pp.
462-463,
1964.
Table 3-10 Aroma and Structure Classification of Browned Flavor Compounds
Aromas:
Structure:
Examples of
compounds:
Burnt (pungent, empyreumatic)
Polycarbonyls(a,p-Unsat'd
aldehydes-C:O-C:0-=C-CHO)
I
Glyoxal
Pyruvaldehyde
Diacetyl
Mesoxalic dialdehyde
Acrolein
Crotonaldehyde
Variable (aldehydic, ketonic)
Monocarbonyls
(R-CHO,
R-C:0-CH
3
)
Strecker aldehydes
lsobutyric
Isovaleric

Methiona!
2-Furaldehydes
2-Pyrrole
aldehydes
C
3
-C
6
Methyl ketones
Source:
From J.E. Hodge, FD. Mills, and B.E.
Fisher,
Compounds of Browned Flavor from
Sugar-Amine
Reac-
tions,
Cereal
Sd.
Today,
Vol. 17, pp.
34-40,
1972.
( MELANOIDINS )
Polymer
Co
-
Polymer
Polymer
AV-containing
compounds

-H
2
O
(
Some
cyciisotco)
Furfurals
Unsaturatcd
osones
D«oxyoson«s
other
carbonyt
compounds
Monofcttose
-ammo
compound
ALOOSE
AHINO
COMPOUND
Atdosylomino
compounds
Unsoturated
car bony
I
compounds
Dikctosc-amino
compound
series,
glycine is the most reactive. Longer
and more complex

substituent
groups reduce
the rate of browning. In the
co-amino
acid
series,
browning rate increases with increas-
ing chain length.
Ornithine
browns more rap-
idly than lysine. When the reactant is a
protein, particular sites in the molecule may
react faster than others. In proteins, the e-
amino group of lysine is particularly vulnera-
ble to attack by aldoses and ketoses.
Moisture content is an important factor in
influencing the rate of the browning
reaction.
Browning occurs at low temperatures and
intermediate moisture content; the rate in-
creases with increasing water content. The
rate is extremely low below the glass transi-
tion temperature, probably because of lim-
ited diffusion (Roos and Himberg 1994;
Roos et
al.
1996a, b).
Methods of preventing browning could
consist of measures intended to slow reaction
rates,

such as control of moisture, tempera-
ture,
or pH, or removal of an active interme-
diate. Generally, it is easier to use an in-
hibitor. One of the most effective inhibitors
of browning is sulfur dioxide. The action of
sulfur dioxide is unique and no other suitable
inhibitor has been found. It is known that
sulfite
can combine with the carbonyl group
of an aldose to give an addition compound:
NaHSO
3
+ RCHO
-> RCHOHSO
3
Na
Time
(minutes).
Figure 3-14 Effect of Temperature on the Reaction Rate of
D-Glucose
with DL-Leucine.
Source:
From
G. Haugard, L.
Tumerman,
and A. Sylvestri, A Study on the Reaction of Aldoses and Amino Acids,
/.
Am. Chem.
Soc.,

Vol. 73, pp.
4594-4600,
1951.
Milhmole
of
DL
-
leucme
per ml
However, this reaction cannot possibly
account for the inhibitory effect of
sulfite.
It
is thought that sulfur dioxide reacts with the
degradation products of the amino sugars,
thus preventing these compounds from con-
densing into
melanoidins.
A serious draw-
back of the use of sulfur dioxide is that it
reacts with thiamine and proteins, thereby
reducing the nutritional value of foods. Sul-
fur dioxide destroys thiamine and is there-
fore not permitted for use in foods containing
this vitamin.
CHEMICAL
CHANGES
During processing and storage of foods, a
number of chemical changes involving pro-
teins may occur (Hurrell 1984). Some of

these may be desirable, others undesirable.
Such chemical changes may lead to com-
pounds that are not hydrolyzable by intesti-
nal enzymes or to modifications of the
peptide side chains that render certain amino
acids unavailable. Mild heat treatments in the
presence of water can significantly improve
the protein's nutritional value in some cases.
Sulfur-containing amino acids may become
more available and certain
antinutritional
fac-
tors such as the trypsin inhibitors of soybeans
may be deactivated. Excessive heat in the
absence of water can be detrimental to pro-
tein quality; for example, in fish proteins,
tryptophan,
arginine, methionine, and lysine
may be damaged. A number of chemical
reactions may take place during heat treat-
ment including decomposition, dehydration
of serine and threonine, loss of sulfur from
cysteine, oxidation of cysteine and methio-
Time
(minutes).
Figure 3-15 Effect of pH on the Reaction Rate of
D-Glucose
with
DL-Leucine.
Source: From G.

Haugard,
L.
Tumerman,
and A. Sylvestri, A Study on the Reaction of Aldoses and Amino Acids, J.
Am.
Chem.
Soc.,
Vol. 73, pp. 4594-4600, 1951.
Millimole
of
OL-leucme
per
ml.
nine,
cyclization of glutamic and aspartic
acids and threonine
(Mauron
1970; 1983).
The nonenzymic browning, or Maillard,
reaction causes the decomposition of certain
amino acids. For this reaction, the presence
of a reducing sugar is required. Heat damage
may also occur in the absence of sugars.
Bjarnason
and Carpenter (1970) demon-
strated that the heating of bovine plasma
albumin for 27 hours at
115
0
C

resulted in a
50 percent loss of cystine and a 4 percent
loss of lysine. These authors suggest that
amide-type bonds are formed by reaction
between the e-amino group of lysine and the
amide groups of asparagine or glutamine,
with the reacting units present either in the
same peptide chain or in neighboring ones
(Figure
3-16).
The Maillard reaction leads to
the formation of brown pigments, or
mel-
anoidins, which are not well defined and
may result in numerous flavor and odor com-
pounds. The browning reaction may also
result in the blocking of lysine. Lysine
becomes unavailable when it is involved in
the
Amadori
reaction, the first stage of
browning. Blockage of lysine is nonexistent
in pasteurization of milk products, and is at
O
to 2 percent in UHT sterilization, 10 to 15
percent in conventional sterilization, and 20
to 50 percent in roller drying
(Hurrell
1984).
Some amino acids may be oxidized by

reacting with free radicals formed by
lipid
oxidation. Methionine can react with a lipid
peroxide to yield methionine sulfoxide. Cys-
teine can be decomposed by a lipid free radi-
cal according to the following scheme:
The decomposition of unsaturated fatty acids
produces reactive carbonyl compounds that
may lead to reactions similar to those
involved in nonenzymic browning. Methio-
nine can be oxidized under aerobic condi-
tions in the presence of
SO
2
as follows:
R-S-CH
3
+
2SO
3
=
->
R-SO-CH
3
+
2SO
4
=
This reaction is catalyzed by manganese ions
at pH values from 6 to 7.5.

SO
2
can also
react with cystine to yield a series of oxida-
tion products. Some of the possible reaction
products resulting from the oxidation of sul-
fur amino acids are listed in Table
3-11.
Nielsen et
al.
(1985) studied the reactions
between protein-bound amino acids and oxi-
dizing lipids. Significant losses occurred of
the amino acids lysine, tryptophan, and histi-
dine.
Methionine was extensively oxidized
to its sulfoxide. Increasing water activity in-
creased losses of lysine and tryptophan but
had no effect on methionine oxidation.
Alkali treatment of proteins is becoming
more common in the food industry and may
result in several undesirable reactions. When
cystine is treated with calcium hydroxide, it
is transformed into
amino-acrylic
acid, hy-
drogen
sulfide,
free sulfur, and
2-methyl

thia-
zolidine-2,
4-dicarboxylic
acid as follows:
Alanine
This can also occur under alkaline condi-
tions,
when cystine is changed into amino-
Cysteine
•*- Pyruvic
acid
(Thiazolidinc)
Figure
3-16
Formation
of
Amide-Type Bonds
from
the
Reaction
Between
£-amine
Groups
of
Lysine
and
Amide Groups
of
Asparagine
(n

=
1)
Glutamine
(n = 2).
Source:
From
J.
Bjarnason
and
K. J.
Carpenter,
Mechanisms
of
Heat
Damage
in
Proteins.
2
Chemical
Changes
in
Pure
Proteins,
Brit.
J.
Nutr.,
Vol. 24, pp.
313-329,
1970.
acrylic acid and

thiocysteine
by a
(3-elimina-
tion mechanism, as
follows:
group of lysine to yield
lysmoalanine
(Zieg-
ler
1964) as shown:
NH
2
-CH-(CH
2
)
4
-NH
2
+
CH
2
=
C-COOH
»•
COOH
NH
2
NH
2
-CH-(CH

2
)
4
-NH-CH
2
—CH-COOH
COOH
NH
2
Lysinoalanine formation is not restricted to
alkaline
conditions—it
can also be formed
by prolonged heat treatment. Any factor
favoring lower pH and less drastic heat treat-
ment will reduce the formation of lysinoala-
nine.
Hurrell
(1984) found that dried whole
milk and UHT milk contained no lysmoala-
nine and that evaporated and sterilized milk
contained 1,000 ppm. More severe treatment
with alkali can decompose arginine into
orni-
thine and urea.
Ornithine
can combine with
dehydroalanine in a reaction similar to the
one giving lysinoalanine and, in this case,
ornithinoalanme

is formed.
Treatment of proteins with ammonia can
result in addition of ammonia to dehydroala-
nine to yield
(3-amino-alanine
as follows:
CH
2
=C-COOH-HNH
3
NH
2
>
NH
2
-CH
2
—CH-COOH
I
NH
2
Light-induced oxidation of proteins has
been shown to lead to off-flavors and destruc-
tion of essential amino acids in milk. Patton
(1954) demonstrated that sunlight attacks
methionine
and converts it into methional
((3-
methylmercaptopropionaldehyde),
which can

cause a typical sunlight off-flavor at a level of
0.1
ppm. It was later demonstrated by Finley
and Shipe (1971) that the source of the light-
induced off-flavor in milk resides in a low-
density lipoprotein fraction.
Ammo-acrylic acid (dehydroalanine) is very
reactive and can combine with the E-amino
Table
3-11
Oxidation Products of the Sulfur-
Containing Amino
Acids
Name
Formula
Methionine
R-S-CH
3
Sulfoxide
R-SO-CH
3
Sulfone
R-SO
2
-CH
3
Cystine
R-S-S-R
Disulfoxide
R-SO-SO-R

Disulfone
R-SO
2
-SO
2
-R
Cysteine
R-SH
Sulfenic
R-SOH
Sulfinic
R-SO
2
H
Sulfonic
(or
R-SO
3
H
cysteic
acid)
Proteins react with polyphenols such as
phenolic acids,
flavonoids,
and tannins,
which occur widely in plant products. These
reactions may result in the lowering of avail-
able lysine, protein digestibility, and biolog-
ical value
(Hurrell

1984).
Racemization is the result of heat and alka-
line treatment of food proteins. The amino
acids present in proteins are of the L-series.
The racemization reaction starts with the
abstraction of an
a-proton
from an amino
acid residue to give a negatively charged pla-
nar carbanion. When a proton is added back
to this optically inactive intermediate, either
a D- or L-enantiomer may be formed (Mas-
ters and Friedman 1980). Racemization leads
to reduced digestibility and protein quality.
FUNCTIONAL
PROPERTIES
Increasing emphasis is being placed on
isolating proteins from various sources and
using them as food ingredients. In many
applications functional properties are of
great importance. Functional properties
have been defined as those physical and
chemical properties that affect the behavior
of proteins in food systems during process-
ing,
storage, preparation, and consumption
(Kinsella
1982). A summary of these proper-
ties is given in Table 3-12.
Even when protein ingredients are added

to food in relatively small amounts, they may
significantly influence some of the physical
properties of the food. Hermansson (1973)
found that addition of 4 percent of a soybean
protein isolate to processed meat signifi-
cantly affected firmness, as measured by
extrusion force, compression work, and sen-
sory evaluation.
The emulsifying and foaming properties of
proteins relate to their adsorption at inter-
faces and to the structure of the protein film
formed there (Mitchell 1986). The emulsify-
ing and emulsion stabilizing capacity of pro-
tein meat additives is important to the
production of sausages. The emulsifying
properties of proteins are also involved in the
production of whipped toppings and coffee
whiteners. The whipping properties of pro-
teins are essential in the production of
whipped toppings. Paulsen and Horan (1965)
determined the functional characteristics of
edible soya flours, especially in relation to
their use in bakery products. They evaluated
the measurable parameters of functional
properties such as water dispersibility, wetta-
bility, solubility, and foaming characteristics
as those properties affected the quality of
baked products containing added soya flour.
Some typical functional properties of food
proteins are listed in Table

3-13.
Surface
Activity
of
Proteins
Proteins can act as surfactants in stabiliz-
ing emulsions and foams. To perform this
function proteins must be amphiphilic just
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